Protein glycosylation is an essential post-translational modification in eukaryotes. The objectives of this proposal are to determine the structure, function and catalytic mechanisms of two distinct enzyme families required for glycoprotein biosynthesis. The first family consists of Class I al,2-mannosidases that have been conserved through eukaryotic evolution from yeast to man. These enzymes are essential for the formation of complex N-glycans on mammalian glycoproteins and therefore are potential targets for the development of antimetastatic and antiviral agents. Large quantities of the catalytic domain of the al,2-mannosidase from Saccharomyces cerevisiae that trims Man9GlcNAc2 to Man8GlcNAc2 have been produced in Pichia pastoris. The three-dimensional structure and catalytic mechanism of this enzyme will be determined using a combination of biochemical approaches, mutagenesis and x-ray crystallography. The other group of enzymes belong to the KRE2/MNTJ Saccharomyces cerevisiae gene family and includes al,2-mannosyltransferases that participate in both 0-and N-linked yeast glycan biosynthesis. There are nine members of this family some of which have overlapping specificities that collectively are essential for viability and for yeast cell wall mannan synthesis. Since they do not exist in mammalian cells, these mannosyltransferases are potential targets for the development of antifungal agents. Large quantities of the catalytic domain of two members of this family have been produced in Pichia pastoris. One of these, Ktrlp, will be purified for structure-function studies. The location and importance of the disulfide bonds and free sulfhydryl groups will be determined by peptide analysis and mutagenesis. The location of the GDPH mannose binding site will be identified by photoaffinity labeling, and attempts will be made to crystallize the enzyme. Determination of the structure and catalytic mechanism of one member of each family will serve as model for the homologous enzymes. Another project is to determine the mechanisms of localization of the yeast al,2-mannosidase, a resident protein of the endoplasmic reticulum (ER) that does not contain any of the known signals implicated in ER targeting. The role of its transmembrane domain will be studied using chimeric constructs and mutagenesis. Co-immunoprecipitation experiments will be done to determine the possible role of oligomerization in ER retention.